The human milk fat globule (HMFG) has been used extensively as a source of antigenic material for the preparation of both polyclonal and monoclonal antibodies that have found widespread use in the diagnosis of breast cancer, as well as in the study of the breast epithelial cell surface and the processing of its antigenic components.
Polyclonal antiserum was originally prepared, that after appropriate absorptions with non-breast tissue was found to identify surface antigens of human mammary epithelial cells (HME-Ags). This antiserum (anti-HME) had a high specificity for normal breast epithelial cells and breast carcinomas. It identified mainly three components of the human milk fat globule which had molecular weights of 150 kDa, 70 kDa, and 46 kDa, respectively.
Monoclonal antibodies were first made against the HMFG in 1980. These antibodies were applied to identify a hitherto unknown component of the breast epithelial cell surface, a large molecular weight mucin-like glycoprotein, that was named non-penetrating glycoprotein (NPGP). This latter component appears to be extremely antigenic in the mouse. The vast majority of monoclonal antibodies prepared against HMFG as well as breast tumors have been found to have specificity against different epitopes of this mucin complex. Less frequently, monoclonal antibodies have been prepared against the 70 kDa and 46 kDa components of the HMFG.
The reason for the high immunogenicity of NPGP has recently been elucidated by the characterization of cDNA clones selected from a kgtll breast cell library using both polyclonal and monoclonal antibodies against the mucin. These cDNA clones consist of large arrays of highly conserved 60 bp tandem repeats. The resulting 20 amino acid repeat contains epitopes for several anti-mucin antibodies.
The repeat is apparently unstable at the genomic level. This may account for the observed polymorphism seen at the gene, RNA and protein levels for this high molecular weight mucin. An initial report on cDNA cloning of the mucin product suggested that the core protein had a molecular weight of about 68 kDa. However, the mRNA was found to be large enough to code for proteins from about 170 kDa to 230 kDa. More recently, using milder deglycosylation methods, a core protein was identified having a molecular weight of about 200 kDa.
Attention has also been devoted to the study and use of the NPGP mucin complex, largely as a result of its high immunogenicity. Thus, a large number of monoclonal antibodies were prepared against it. However, the smaller components of HMFG also appear to be important molecules on the surface of breast epithelial cells. They have a breast specificity as demonstrated by the anti-HME antibodies.
The 46 kDa and 70 kDa HME antigens are found in serum of breast cancer patients and thus can be used as markers for breast cancer in serum assays. In addition, the 70 kDa component has been found to co-purify with the intact mucin complex and has been reported to be associated with the NPGP mucin complex by means of disulfide bonds, making it a possible linker protein of this surface mucin complex.
Few monoclonal antibodies have been prepared against the smaller components of the system. The mucin molecule is apparently more antigenic because of its internally repeated structure. The 46 kDa component of HMFG has been found in the serum of breast cancer patients. Using monoclonal antibodies against the 46 kDa antigen, circulating immune complexes were found in breast cancer patients and an increase in the circulating 46 kDa antigen was found to be associated with increased tumor burden. The structure of the mucin glycoprotein has recently been determined by cDNA cloning and a partial sequence has been reported for the 70 kDa antigen.
Very little, however, is known about the structure of the 46 kDa antigen and its function, along with the other membrane components, in the normal epithelial cell membrane, milk formation, and breast tumorigenesis. Up to the present time neither the sequences of the about 46 kDalton polypeptide component nor the DNA and RNA encoding it were known.